It is well known that steroid molecules can be transformed by microorganisms (W. Charney, H. L. Herzog, Microbial Transformations of Steroids). Many processes using microbiological transformations of steroids have been developed, to replace cumbersome and expensive chemical processes. A cheap source for the preparation of useful steroid intermediates are the abundantly available sterols, for example cholesterol and sitosterol. Microorganisms have been selected which are able to use these steroids as a carbon source. In several patent publications, e.g. Dutch patent applications NL 6513718 and NL 6705450, the microbiological degradation of the C-17-side chain with the preservation of the steroid nucleus is described. With specific inhibitor compounds and later also with specially developed mutants, as described in U.S. Pat. Nos. 3,684,657, 3,759,791 or 4,345,029, it was possible to obtain in high yield androst-4-ene-3,17-dione and androsta-1,4-diene-3,17- dione, which are starting compounds for the synthesis of therapeutically useful steroids.
Among the microbiologically prepared steroids 9.alpha.-hydroxy-17-keto steroids are important because they are valuable starting compounds for the preparation of corticosteroids (J. Org. Chem. (1979) 44, 1582). The necessary introduction in prospective corticosteroids of a hydroxyl group on C-11 and optionally of a halogen atom on C-9 can be carried out by easy, well established chemical reactions, departing from this class of compounds.
A preferred compound of this type is 9.alpha.-hydroxyandrost-4-ene-3,17-dione, originally described in J. Amer. Chem. Soc. 80 (1958), 6148. It possesses anti-androgenic and anti-oestrogenic activity, but it is mainly used as an intermediate in the synthesis of therapeutically valuable corticosteroids (see e.g. European patent application EP-A-0263569).
In the following patent publications several methods are disclosed for obtaining 9.alpha.-hydroxy-17-keto steroids, particularly 9.alpha.-hydroxyandrost-4-ene-3,17-dione:
a. Microbiological introduction of the 9.alpha.-hydroxyl group starting from androst-4-ene-3,17-dione using e.g. a microorganism of the genus Nocardia (U.S. Pat. No. 4,397,947) or a Corvnespora cassicola strain (European patent application EP-A-0027829).
b. U.S. Pat. No. 3,759,791 (examples 6 and 7) describes the presence of 9.alpha.-hydroxyandrost-4-ene-3,17-dione in the broth after the fermentation of sterols with a Mycobacterium strain. According to U.S. Pat. No. 4,035,236 Mycobacterium fortuitum NRRL B-8119 is able, though in a rather low yield, to degrade the C-17-side chain of sterols with the simultaneous introduction of the 9.alpha.-hydroxyl group. Besides the low yield, which is caused predominantly by the gradual further degradation of the desired product during incubation, a drawback of this process is that the microorganism is considered to be an opportunistic pathogen.
c. East German patent DD 232167 discloses the preparation of 9.alpha.-hydroxyandrost-4-ene-3,17-dione by the strain Mycobacterium fortuitum N10 (ZIMET 10849). According to a special feature the yield is raised to 40-50% by adding a finely dispersed, hydrophobic organic polymer to the fermentation medium.
d. The preparation of 9.alpha.-hydroxyandrost-4-ene-3,17-dione departing from sterols by using a Mycobacterium strain is also described in Japanese patent application JP 55/85397, but only a low yield is reported.
e. According to British patent application GB 2197869, published Jun. 2, 1988, production of 9.alpha.-hydroxyandrost-4-ene-3,17-dione is effected by sterol fermentation using a strain of the novel Mycobacterium roseum species. From 100 g sterol 28.5 g product is obtained.